Plant cell walls are composed of crystalline cellulose entangled with hemi-cellulose and lignin, forming a complex matrix rendering plant biomass largely inaccessible to cellulolytic enzymes in the native state. Current methods of lignocellulosic biofuel production typically involve disrupting plant cell walls using high temperatures and/or corrosive chemicals to liberate the polysaccharides and generate a product that is more accessible to hydrolytic saccharification. These pretreatments are costly, inefficient and, in certain cases, are environmentally toxic. It is, therefore, necessary to improve pretreatment methods.
Ionic liquids (ILs) represents a promising solution to the problem of recalcitrant biomass. ILs are nonvolatile salts, typically with melting points under 100° C., and some ILs can efficiently solubilize cellulose, hemicellulose and lignin from plant biomass under moderate temperatures1-6. The regeneration of cellulose from ILs can be achieved by adding an anti-solvent, such as water or ethanol, into the solution7-9. ILs can be recycled for new rounds of pretreatment. It has been shown that regenerated cellulose after IL pretreatment has reduced crystallinity, and is thus easier for cellulolytic enzymes to access10-11.
Several improvements are needed in the ionic liquid pretreatment process technology before it is cost effective with other pretreatments that are based on the pulp and paper processing technologies that utilize dilute acids and bases. One of the most important areas for cost reduction is reducing the number of washes required after IL pretreatment. Unfortunately, commercial fungal cellulases are inhibited by some ILs8, 12-13 and, therefore, require extensive washing after IL pretreatment. Therefore, it is crucial to identify IL-resistant enzymes for digesting cellulose in the presence of ionic liquids to decrease the number of washes required and increase the yields of monomeric sugars.
It has been suggested that ILs inhibit enzymatic activity by disrupting hydrogen bonding and hydrophobic interactions and depriving the water hydration shell of the protein14-18. This is similar to the denaturing effect caused by salt on mesophilic proteins. Although it's not clear if salt and ionic liquids denature proteins in identical ways, both create an environment characterized by low-water activity and high ionic strength14, 19. Microbes living in extremely high salt environments can possess a cytoplasm containing >3 M salt. Accordingly, such organisms have evolved a unique mechanism to compete with salt for water. In high salt concentrations, proteins contain an excessive number of negatively charged acidic amino acids on their surface, while at the same time having only few basic amino acids and a low hydrophobic amino acid content20-23. Among these, the negative charges are the most prominent feature24-27 and are thought to keep the protein soluble in a high salt solution either by forming a hydrated ion network with cations or by preventing the formation of protein aggregation through electrostatic repulsive charges at the protein surface25, 28-31. Theoretically, positive charges on protein surface may have similar effect on protein stability in high salt environments as negative charges. Yet, in nature, majority of the halophilic proteins are enriched with acidic amino acids on the protein surface, suggesting that negatively charged proteins are under positive selection in halophilic microorganisms. In addition, reduced surface area is also important for the protein to remain folded and require less water to form a hydration shell. All of these salt adaptation strategies could be used for enzymes to resist ILs.